Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Clorobiocin, an inhibitor of the gyrB subunit of DNA gyrase, was used for the curing of some Escherichia coli plasmids. Of the plasmids studied, ampicillin resistant R28K and a miniplasmid derived from R1drd-19 were effectively eliminated. We also succeeded in eliminating the ColA factor from E. coli strain B834(pBS103), which was resistant to the effect of currently used curing agents. Although a derivative of ColE1-pBR322 was effectively cured by clorobiocin, the ColE1 plasmid was resistant to its effect. The ColV plasmid determining virulence was effectively eliminated.
Mol Gen Genet 1982
PMID:Curing effect of clorobiocin on Escherichia coli plasmids. 705 Jun 23

An in frame gene fusion containing the coding region for mature beta-lactamase and the 3'-end of hylA encoding the haemolysin secretion signal, was constructed under the control of a lac promoter. The resulting 53 kDa hybrid protein was specifically secreted to the external medium in the presence of the haemolysin translocator proteins, HlyB and HlyD. The specific activity of the beta-lactamase portion of the secreted protein (measured by the hydrolysis of penicillin G), approximately 1 U/microgram protein, was close to that of authentic, purified TEM-beta-lactamase. This is an important example of a hybrid protein that is enzymatically active, and secreted via the haemolysin pathway. Previous studies have indicated that haemolysin is secreted directly into the medium, bypassing the periplasm, to which beta-lactamase is normally targeted. This study indicated, therefore, that normal folding of an active beta-lactamase, can occur, at least when fused to the HlyA C-terminus, without the necessity of entering the periplasm. Despite the secretion of approximately 5 micrograms/ml levels of the active beta-lactamase fusion into the medium, there was maximally only a 50% detectable increase in the LD50 for resistance to ampicillin at the individual cell level. This result suggests that, normally, resistance to ampicillin requires a high concentration of the enzyme close to killing targets, i.e. in the periplasm, in order to achieve significant levels of protection.
Mol Gen Genet 1995 Nov 15
PMID:Secretion of active beta-lactamase to the medium mediated by the Escherichia coli haemolysin transport pathway. 750 Sep 46

Urease is a virulence determinant, a taxonomic and diagnostic marker, and immunogen for Helicobacter pylori, an aetiologic agent of gastritis and peptic ulceration. This enzyme requires Ni2+ ions in the active site for successful hydrolysis of urea. When expressed in Escherichia coli, recombinant urease is only weakly active unless urease structural subunits are overexpressed, exogenous NiCl2 is added, and the host strain is grown in medium that does not chelate free Ni2+. As wild-type H. pylori does not require such conditions for very high levels of urease expression, we reasoned that additional genes were required to accumulate the metal ion. To isolate such genes, E. coli SE5000 (pHP808), which carries the H. pylori urease gene cluster, was complemented with a lambda ZAP-derived plasmid library of the H. pylori chromosome. One of 1000 ampicillin-resistant clones, plated onto urea segregation agar, produced detectable urease. Urease activity of this co-transformant, grown in Luria broth containing 1 microM NiCl2, was 36 mumol NH3 min-1 mg-1 protein. Urease-enhancing activity, which is not directly linked to the urease gene cluster, was localized by subcloning and nucleotide sequencing. The largest open reading frame, designated nixA, predicted a polypeptide of 34,317 Da that displayed characteristics of an integral membrane protein. In vitro transcription-translation of nixA sequences yielded a polypeptide estimated to be 32 kDa in size. An in-frame Bal31 deletion within nixA abolished urease-enhancing activity. At 50 nM NiCl2, E. coli containing the nixA clone transported 1250 +/- 460 pmol Ni2+ min-1 10(-8) cells, whereas the vector control transported only 140 +/- 85 pmol Ni2+ min-1 10(8) cells, i.e. significantly less (P = 0.01). We conclude that NixA confers upon E. coli a high-affinity nickel-transport system (KT = 11.3 +/- 2.4 nM; Vmax = 1750 +/- 220 pmol Ni2+ min-1 10(-8) cells) and is necessary for expression of catalytically active urease, regardless of growth conditions.
Mol Microbiol 1995 Apr
PMID:Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions. 765 Nov 42

Under superhelical stress, oligopurine-oligopyrimidine mirror-repeat sequences are able to adopt H-DNA conformations where a triple-helical and a single-stranded structure co-exist. We have previously shown that a benzo[e]pyridoindole derivative (BePI), an antitumor drug interacting more tightly with triplex than with duplex DNA, strongly stabilizes intermolecular triple helices formed upon binding of homopyrimidine oligonucleotides to the major groove of double-stranded DNA at oligopurine-oligopyrimidine sequences. Here we show that an intramolecular triple helix is also strongly stabilized by this ligand. In vitro elongation performed by different DNA polymerases (bacteriophage T7, Escherichia coli or Taq polymerase) could be irreversibly inhibited by the H-DNA structure in the presence of BePI. A mirror-repeat polypurine-polypyrimidine sequence inserted between the E. coli beta-lactamase gene (conferring ampicillin resistance) and its bla promoter strongly inhibited transcription of the beta-lactamase gene in vivo. In the absence of supercoiling, transition to the H-conformation did not occur, but BePI stabilized the H-DNA structure induced by supercoiling as shown by chemical probes (chloroacetaldehyde). The results presented here open a new field of investigation for antitumor agents targeted to a novel class of genetic structures able to regulate gene expression.
J Mol Biol 1995 Apr 14
PMID:Triple-helix specific ligands stabilize H-DNA conformation. 772 37

The hns gene is a member of the cold-shock regulon, indicating that the nucleoid-associated, DNA-binding protein H-NS plays an important role in the adaptation of Escherichia coli to low temperatures. We show here that the ability to cope efficiently with a cold environment (12 degrees C and 25 degrees C) is strongly impaired in E. coli strains carrying hns mutations. Growth inhibition is much more pronounced in strains carrying the hns-206 allele (an ampicillin resistance cassette inserted after codon 37) than in those carrying the hns-205 mutation (a Tn10 insertion located in codon 93). A protein fragment (H-NS*) is synthesized in strains carrying the hns-205::Tn10 mutation, suggesting that this truncated polypeptide is partially functional in the cold adaptation process. Analysis of the growth properties of strains harbouring four different low-copy-number plasmid-encoded hns' genes that result in the production of C-terminally truncated H-NS proteins supports this proposal. H-NS* proteins composed of 133, 117 or 94 amino-terminal amino acids partially complemented the severe cold-sensitive growth phenotype of the hns-206 mutant. In contrast, synthesis of a truncated H-NS protein with only 75 amino-terminal amino acids was insufficient to restore growth at low temperature.
Mol Gen Genet 1994 Oct 28
PMID:The nucleoid-associated DNA-binding protein H-NS is required for the efficient adaptation of Escherichia coli K-12 to a cold environment. 781 34

We selected cDNA plasmid clones that corrected the temperature-sensitive phenotype of Escherichia coli strain JC201, which is deficient in 1-acyl-sn-glycerol-3-phosphate acyltransferase activity. A plasmid-based maize endosperm cDNA library was used for complementation and a plasmid that enabled the cells to grow at 44 degrees C on ampicillin was isolated. Addition of this plasmid (pMAT1) to JC201 restored 1-acyl-sn-glycerol-3-phosphate acyltransferase activity to the cells. Total phospholipid labelling showed that the substrate for the enzyme, lysophosphatidic acid, accumulated in JC201 and was further metabolised to phosphatidylethanolamine in complemented cells. Membranes isolated from such cells were able to convert lysophosphatidic acid to phosphatidic acid in acyltransferase assays. The cDNA insert of pMAT1 contains one long open reading frame of 374 amino acids which encodes a protein of relative molecular weight 42,543. The sequence of this protein is most similar to SLC1, which is thought to be able to acylate glycerol at the sn-2 position during synthesis of inositol-containing lipids. Homologies between the SLC1 protein, the 1-acyl-sn-glycerol-3-phosphate acyltransferase of E. coli (PlsC) and the maize ORF were found with blocks of conserved amino acids, whose spacing was conserved between the three proteins, identifiable.
Plant Mol Biol 1994 Oct
PMID:Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a protein which has similarities to other acyltransferases. 794 71

The use of new molecular typing methods for the characterization of Haemophilus influenzae strains is reported. Sixty-four isolates of H. influenzae originating from different types of infection and obtained from eight hospitals across Canada were first analysed for restriction polymorphism. Chromosomal DNA fragments generated by two different combinations of restriction endonucleases were electrophoresed and transferred to nylon membranes before hybridization with a species specific 32P-labelled DNA fragment (5 kb) used as a probe. The combinations Bg/II/PstI led to 11 typing groups (A-K) and BamHI/Bg/II/PstI to 14 sub-groups, respectively. Most of the isolates retrieved from cerebrospinal fluids (10/13; 76.9%) were classified in two groups (A and B) and two sub-groups. Isolates from respiratory tract infections were mostly found in groups C and E (24/32; 75.0%), and divided into seven sub-groups. Selected ampicillin-resistant, beta-lactamase-negative strains were also found in groups C and E (11/14; 78.6%). Isolates from conjunctivitis and acute otitis media were classified in various groups. All biotypes (I-VIII) and serotypes (none, a-f) were spread among the typing groups although biotype I prevailed in groups A, B, and G; II in group E (sub-group 6); and III in group C. A PCR approach derived from the typing system was also tested. A set of 25-mer primers was selected from the 5-kb DNA probe for the amplification of a 317-bp region. This set of primers was used concomitantly in a PCR multiplex assay with a set of primers selected from the nucleotide sequence of the gene encoding the H. influenzae P1 protein. This multiplex assay was also able to discriminate the clonal origin of some H. influenzae strains because size polymorphism was observed in PCR products. The PCR approach was then used to determine the genetic relatedness of H. influenzae strains found persistently in sputa of some patients with cystic fibrosis. Genetically related strains could be isolated from some patients even after antibiotherapy and months between visits, whereas other patients showed distinct strains. In summary, our typing system is able to provide new characteristics for strains having identical biotype or serotype. The rapid PCR alternative may prove useful for specific epidemiological and strain-tracking studies.
Mol Cell Probes 1994 Feb
PMID:Molecular typing of Haemophilus influenzae using a DNA probe and multiplex PCR. 802 5

In order to understand how TEM-1 beta-lactamase substrate specificity can be altered by mutation, amino acid residues 161 through to 170 were randomly mutagenized to sample all possible amino acid substitutions. The 161-170 region includes a portion of an omega loop structure, which is involved in the formation of the active-site pocket. The percentage of random sequences that provide bacterial resistance to either ampicillin or to the extended-spectrum cephalosporin ceftazidime was determined. It was found that the sequence requirements for wild-type levels of ampicillin resistance are much more stringent than the sequence requirements for ceftazidime resistance. Surprisingly, more than 50% of all amino acid substitutions in the 161-170 region result in levels of ceftazidime resistance at least three times greater than wild type. In addition, by increasing the level of the selection for ceftazidime resistance, substitutions that result in a greater than 100-fold increase in ceftazidime resistance were identified. Characterization of altered beta-lactamase enzymes indicated that while their catalytic efficiency (kcat/Km) for ceftazidime hydrolysis is higher, the enzymes are poorly expressed relative to wild-type TEM-1 beta-lactamase.
Mol Microbiol 1994 Apr
PMID:Evolution of antibiotic resistance: several different amino acid substitutions in an active site loop alter the substrate profile of beta-lactamase. 805 47

The crystal structure of beta-lactamase from Staphylococcus aureus inactivated by p-nitrophenyl[[N-(benzyloxycarbonyl)amino]methyl]phosphonate, a methylphosphonate monoester monoanion inhibitor, has been determined and refined at 2.3 A resolution. The structure reveals a tetrahedral phosphorus covalently bonded to the O gamma atom of the active site serine, Ser70. One of the oxygen atoms bonded to phosphorus is located in the oxyanion hole formed by the two main-chain nitrogen atoms of Ser70 and Gln237, and the second bonded oxygen is solvated. The (benzyloxycarbonyl)aminomethyl group is oriented towards the active site gully such that the peptide group forms compensating electrostatic interactions with polar groups on the enzyme. The benzyl group forms a hydrophobic interaction with Ile239 and an aromatic-aromatic edge-to-face interaction with Tyr105, which has undergone a conformational transition relative to the native structure. The mode of binding supports the proposal that on reaction with the enzyme, the phosphonate generates a structure analogous to the tetrahedral transition state/intermediate associated with the acylation step of a normal substrate. The disposition of the phosphonyl group in this complex is the same as that of the corresponding phosphoryl group in the complex resulting from the inhibition of trypsin by diisopropylphosphofluoridate. The structure is consistent with a mechanism of inactivation that follows an associative pathway, proceeding via a transition state/intermediate in which phosphorus is penta-co-ordinated, forming a trigonal bipyramidal geometry with the phosphonyl donor (p-nitrophenol) and acceptor (Ser70 O gamma atom) in apical positions. A model of this transition state can be accommodated in the active site of beta-lactamase without any steric hindrance. A model of the tetrahedral transition state associated with the acylation step by benzyl penicillin has been derived. Because of the conformational rigidity of the fused rings of penicillin molecules, the orientation of the substrate is fixed once the tetrahedral carbonyl carbon and its ligands are superimposed on the phosphonate group. The outcome is that the carboxylate substituent on the thiazolidine ring forms a salt bridge with Lys234, and the preferred puckering of the ring is that observed in the crystal structure of ampicillin, the so-called "open" conformer.
J Mol Biol 1993 Nov 05
PMID:Structure of a phosphonate-inhibited beta-lactamase. An analog of the tetrahedral transition state/intermediate of beta-lactam hydrolysis. 823 Jan 96

Two isolates of bacterial endosymbionts, GP01 and GM02, were established in cell free medium from haemolymph of the tsetse, Glossina pallidipes and G. morsitans. These microorganisms appear similar to rickettsia-like organisms reported previously from various tsetse species. The 16S rRNA sequence analysis, however, placed them within the gamma subdivision of the Proteobacteria, phylogenetically distinct from most members of the Rickettsiaceae which align with the alpha subdivision. Distinct multiple endogenous plasmids are harboured by GP01 and GM02, suggesting that the two isolates are different. Restriction mapping analysis showed that one of the conserved plasmids is present in high copy number and is at least 80 kb in size. A heterologous plasmid pSUP204, which contains the broad host range oriV replication origin, was used to transfect bacterial cultures. The symbiont GM02 was transformed, and it expressed plasmid encoded resistance to the antibiotics ampicillin, tetracycline and chloramphenicol. Transformation of these symbionts may provide a novel means for expressing anti-parasitic genes within tsetse populations.
Insect Mol Biol 1993
PMID:Genetic transformation and phylogeny of bacterial symbionts from tsetse. 826 90


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>